The present invention relates to the crystal structure of the ACPS/ACP complex, as well as the three-dimensional solution structure of B. subtilis ACP. These structures are critical for the design and selection of potent and selective agents which interact with ACPS and ACP, and particularly, the design of novel antibiotics.
Acyl Carrier Proteins (ACPs) play important roles in a number of biosynthetic pathways that are dependent upon acyl group transfers [1]. They are most often associated with the biosynthesis of fatty acids [2,3], but they are also utilized in the synthesis of polyketide antibiotics [4,5], non-ribosomal peptides [6,7], and of intermediates used in the synthesis of vitamins such as the protein-bound coenzymes, lipoic acid [8] and biotin [9]. The ACP in each of these pathways is composed of 80-100 residues and is either an integrated domain in a larger multi-functional protein (Type I synthase complex) or is a structurally independent protein that is part of a non-aggregated multi-enzyme system (Type II synthase complex). Type I synthases are found in mammals, fungi and certain Mycobacteria while type II ACPs are utilized by plants and most bacteria. The Escherichia coli ACP for fatty acid synthesis has been over-expressed [10] and purified [11,12], and the solution structure has been solved by NMR spectroscopy [13]. The fact that these proteins are essential for the maturation of the organism has led to their investigation as targets for the development of new anti-microbial agents [14-18].
ACPs require post-translational modification for activity. They are converted from an inactive apo-form to an active holo-form by the transfer of the 4xe2x80x2-phosphopantetheinyl (P-pant) moiety of coenzyme A to a conserved serine on the ACP. The xcex2-hydroxy side chain of the serine residue serves as a nucleophilic group attacking the pyrophosphate linkage of CoA. Evidence now suggests [19] that each synthase that is dependent upon P-pant attachment for activation has its own partner enzyme responsible for this attachment.
The post-translational modification of the ACP subunit in the fatty acid synthase is performed by holo-[acyl carrier protein] synthase (hereinafter defined as xe2x80x9cACPSxe2x80x9d; Enzyme Commission No. 2.7.8.7). The best characterized member of the ACPS family is the E. coli ACPS [20]. The enzyme produces holo-ACP by transferring the P-pant moiety to Ser-36 of the E. coli apo-ACP in a magnesium dependent reaction [20] as follows: 
The over-expression and purification of the E. coli ACPS has been described [21] and this protein is classified as a member of a new enzyme superfamily, the phosphopantetheinyl transferases [19]. Based on the size of the proteins, the P-pant transferase superfamily can be roughly divided into two subgroups [22]. Enzymes responsible for modifying the peptidyl carrier protein subunits of non-ribosomal peptide synthetases are good examples for the first subgroup, which are usually xcx9c230 amino acids in size. The structure of one of this subgroup enzymes, the surfactin synthetase activating enzyme Sfp, has been solved recently and it consists of a 2-fold intramolecular pseudosymmetry with the CoA binding site at the interface of the symmetrical fold [22]. ACPS and other enzymes transfering the P-pant group onto domains of the fatty acid synthases are usually smaller, about xcx9c120 residues, and belong to the second subgroup of the P-pant transferase superfamily. The sequence homology between these two subgroups is rather low, about 12-22% between E. coli ACPS and B. subtilis Sfp, for example, although both have been shown to possess P-pant transferase activity. Alignment [19] of some of these proteins show that two regions, residues 5-13 and 54-65 (E. coli ACPS numbering), are highly conserved with five of the residues in these regions identical.
While numerous members of the phosphopantetheinyl transferase superfamily have been identified and sequenced, until the present invention, the crystal structure of ACPS complexed with halo-ACP, and the three dimensional structure of the ACPS/ACP active site has not been determined. Further, prior to the present invention, the solution structure of B. subtilis ACP had not been determined.
The present invention relates to a crystallized complex comprising an acyl carrier protein synthase (ACPS) and an acyl carrier protein (ACP) (hereinafter referred to as xe2x80x9cACPS-ACP complexxe2x80x9d). The invention is further directed to the three dimensional structure of the ACPS-ACP complex, as determined using crystallographic analysis (with or without sedimentation analysis) of the ACPS-ACP complex. Particularly, the invention is directed to the three dimensional structure of the ACP binding site present in ACPS and other ACPS-like P-pant transferases, alone, and as complexed with ACP or other agents that interact with the ACP binding site of said transferases. In addition, the invention is directed to the ACPS binding site on ACP. Identification of the three dimensional structure of the ACP binding site on ACPS and the ACPS binding site on ACP will be valuable for the design of antibiotics and other agents that interfere with P-pant attachment, thereby preventing activation of corresponding carrier proteins.
The invention additionally provides a method for identifying an agent that interacts with any active site of an ACPS-ACP complex, comprising the steps of determining a putative active site of an ACPS-ACP complex from a three dimensional model of the ACPS-ACP complex, and performing various computer fitting analyses to identify an agent which interacts with the putative active site. Again, such agents may act as inhibitors or activators of ACPS-ACP complex activity, as determined by obtaining the identified agent, contacting the same with ACPS-ACP complex, and measuring the agent""s effect on ACPS-ACP complex activity.
In addition, the invention provides a solution comprising B. subtilis ACP having a three dimensional structure defined by the structural coordinates of FIG. 5 and 5-A1 to 5-A15,xc2x1a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 xc3x85. Also provided by the invention is any active site of B. subtilis ACP that is defined by the structural coordinates of FIG. 5 and 5-A1 to 5-A15,xc2x1a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 xc3x85. Further, the present invention provides a method for identifying an agent that interacts with any active site of B. subtilis ACP, comprising the steps of determining a putative active site of ACP from a three dimensional model of the ACP, and performing various computer fitting analyses to identify an agent which interacts with the putative active site. Again, such agents may act as inhibitors or activators of ACP activity, as determined by obtaining the identified agent, contacting the same with ACP, and measuring the agent""s effect on ACP activity.
Yet another aspect of the present invention is a method for identifying an activator or inhibitor of any molecule or molecular complex which comprises an ACP binding site, including any member of the ACPS-like P-pant transferases, comprising the steps of generating a three dimensional model of said molecule or molecular complex using the relative structural coordinates according to FIGS. 3 and 3A-1 to 3A-79 of residues ARG14, MET18, ARG21, GLN22, ARG24, PHE25, ARG28, PHE54, GLU58, ILE68, GLY69, ALA70, SER73 and PHE74 from a first monomer of ACPS, and residue ARG45 from a second monomer of ACPS, or additionally, of residues ASP8, ILE9, THR10, GLU11, LEU12, ILE15, ALA16, SER17, ALA19, GLY20, ALA23, ALA26, GLU27, ILE29, ALA51, LYS57, SER61, LYS62, THR66, GLY67, GLN71, LEU72, GLN75, ASP76, ILE77 and LYS93 from the first monomer of ACPS and residues LEU41, SER42, LYS44, GLU48, GLN83, ASN84, HIS105, THR106 and ALA107 from the second monomer of ACPS, in each casexc2x1a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 xc3x85, and then selecting or designing a candidate activator or inhibitor that interacts with said molecule or molecular complex using computer fitting analyses of interactions between the three dimensional model of the molecule or molecular complex and the candidate activator or inhibitor. The effect of the candidate activator or inhibitor may be evaluated by obtaining the candidate activator or inhibitor, contacting the same with the molecule or molecular complex, and measuring the effect of the candidate activator or inhibitor on molecular or molecular complex activity.
In addition, the present invention provides a method for identifying an activator or inhibitor of any molecule or molecular complex which comprises an ACPS binding site, comprising the steps of generating a three dimensional model of said molecule or molecular complex comprising an ACPS binding site using the relative structural coordinates according to FIGS. 3 and 3A-1 to 3A-79 or FIGS. 5 and 5-A1 to 5-A15 of residues ARG14, LYS29, ASP35, SER36, LEU37, ASP38, VAL40, GLU41, VAL43, MET44, GLU47, ASP48, ILE54, SER55, ASP56, GLU57 and GLU60, or additionally, of residues ASP13, LEU15, PHE28, GLU30, ASP31, LEU32, GLY33, ALA34, VAL39, LEU42, GLU45, LEU46, GLU49, MET52, GLU53, ASP58, ALA59, and LYS61, in each casexc2x1a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 xc3x85, and then selecting or designing a candidate activator or inhibitor that interacts with said molecule or molecular complex using computer fitting analyses of interactions between the three dimensional model of the molecule or molecular complex and the candidate activator or inhibitor. The effect of the candidate activator or inhibitor may be evaluated by obtaining the candidate activator or inhibitor, contacting the same with the molecule or molecular complex, and measuring the effect of the candidate activator or inhibitor on molecular or molecular complex activity. Also provided by the present invention are the activators or inhibitors selected or designed using the above-noted methods.
Still further, the present invention is directed to a method of determining the three dimensional structure of a molecule or molecular complex whose structure is unknown, comprising the steps of first obtaining crystals of the molecule or molecular complex whose structure is unknown, and then generating X-ray diffraction data from the crystallized molecule or molecular complex. The X-ray diffraction data from the molecule or molecular complex is compared with the known three dimensional structures determined from the ACPS-ACP crystals of the present invention, and molecular replacement analysis is used to conform the known three dimensional structures to the X-ray diffraction data from the crystallized molecule or molecular complex.
In addition, the present invention provides the ACP active site of an ACPS-like P-pant transferase, including, but not limited to, an ACPS, comprising the structural coordinates according to FIGS. 3 and 3A-1 to 3A-79 of residues ARG14, MET18, ARG21, GLN22, ARG24, PHE25, ARG28, PHE54, GLU58, ILE68, GLY69, ALA70, SER73 and PHE74 from a first monomer of ACPS, and residue ARG45 from a second monomer of ACPS, in each casexc2x1a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 xc3x85. In another embodiment, the active site may include, in addition to the structural coordinates above, the relative the structural coordinates according to FIGS. 3 and 3A-1 to 3A-79 of residues ASP8, ILE9, THR10, GLU11, LEU12, ILE15, ALA16, SER17, ALA19, GLY20, ALA23, ALA26, GLU27, ILE29, ALA51, LYS57, SER61, LYS62, THR66, GLY67, GLN71, LEU72, GLN75, ASP76, ILE77 and LYS93 from one monomer of ACPS and residues LEU41, SER42, LYS44, GLU48, GLN83, ASN84, HIS105, THR106 and ALA107 from a second monomer of ACPS, in each casexc2x1a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 xc3x85.
Finally, the present invention provides the ACPS active site of ACP, comprising the structural coordinates according to FIGS. 3 and 3A-1 to 3A-79 or FIGS. 5 and 5-A1 to 5-A15 of residues ARG14, LYS29, ASP35, SER36, LEU37, ASP38, VAL40, GLU41, VAL43, MET44, GLU47, ASP48, ILE54, SER55, ASP56, GLU57 and GLU60, in each casexc2x1a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 xc3x85. In another embodiment, the active site may include, in addition to the structural coordinates above, the relative structural coordinates according to FIGS. 3 and 3A-1 to 3A-79 or FIGS. 5 and 5-A1 to 5-A15 of residues ASP13, LEU15, PHE28, GLU30, ASP31, LEU32, GLY33, ALA34, VAL39, LEU42, GLU45, LEU46, GLU49, MET52, GLU53, ASP58, ALA59, and LYS61,xc2x1a root mean square deviation from the backbone atoms of said amino acids of not more than 1.5 xc3x85.